Osteoporosis is a major public health problem, especially in women. It is mainly characterized by low bone mineral density (BMD). Women have much lower BMD than men. Some BMD genes/genomic regions are sex-specific. Menopause is associated with rapid bone loss. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs), are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. The GOAL of this project is to identify genes that are differentially expressed (at mRNA levels) in BMMSCs and PBMs in females with low vs. high BMD and with menopausal status changes. Such genes are expected to be important for variation of female BMD and women health in general. Our preliminary functional genomic studies of PBMs suggested HDC and RUNX1 (NOT the extensively studied RUNX2) genes to be important in determining BMD in humans. Project 2 is built upon this stimulating lead to screen much larger and more powerful samples of both BMMSCs and 3BMs to confirm and extend our preliminary studies and to comprehensively screen genes potentially important for BMD. This project has two inherently related aspects or stages; research in Stage 2 is for exemplifying functional studies to follow the completion of research in Stage 1 and in Projects 1 and 3. STAGE1 (Primary): Whole genome gene differential expression (WGGDE) study. Hypothesis: Changes in the mRNA expression profiles in female BMMSCs and PBMs underlie mechanisms of female BMD variation and are associated with menopause. Specific Goals: To identify genes differentially expressed in BMMSCs and PBMs in women: 1) with high vs. low BMD; 2) before and after menopause, and thus identify genes associated with female BMD and menopause. We will recruit 80 otherwise healthy females and 80 age-matched otherwise healthy males aged 50-55, stratified by discordant BMD values and menopausal status (for females). We will perform bone marrow aspiration and obtain peripheral blood samples. BMMSCs and PBMs will be isolated and total RNA extracted. Microarray profiling experiments and analyses will be performed on females for >40,000 known human genes and ESTs. Differentially expressed genes will be verified by real-time RT-PCR with female samples. These verified genes in females will be examined by real-time RT-PCR with male samples to examine their sex-specificity. STAGE2 (Secondary): Functional studies of molecular mechanisms of candidate genes. As an exploratory example, we will present and perform in-depth functional studies to dissect the mechanisms through which HDC and RUNX1 genes regulate BMD. We hypothesize that HDC and RUNX1 genes are important for osteoclast differentiation and/or bone resorption and HDC is a mediator for RLJNX1 gene in regulating BMD. The results, together with those from Projects 1 and Project 3, will powerfully and efficiently identify genes and some of their functions for female osteoporosis. Identifying genes for human BMD variation, especially for women, is important for 1) gaining insights into the fundamental molecular mechanisms of risk to osteoporosis, 2) discovering new pathways and targets for therapeutic cures; 3) identifying genetically susceptible individuals (by designing diagnostic DMA chip), so that future preventions and interventions can be targeted to and based on individuals' specific genotypes.